The biological glues are adhesive protein concentrates composed of fibrin generated from fibrinogen activated by thrombin and factor XIII in presence of calcium ions. The adhesive power of blood clot, due to it network of polymerized fibrin, has been known for a long time. Fibrin has been used since the beginning of this century as an adhesive and discovered by Bergel in 1909 who recognized it as a physiological gluing substance and moreover ascribed it healing properties. This discovery was immediately followed by Grey's work in 1915 using fibrin tampons to stop brain, liver haemorrhages and in cerebral surgery. However, it is only in 1944 that Cronkite, then Tidrick and Warner used fibrinogen together with thrombin to secure skin graft. But the low concentration of these products did not allow a good quality adhesion nor a lasting effect. Since 1946, owing to important scientific research by E. J. Cohn on the fractionation of plasma proteins, coagulation proteins in particular have been used, and few years later the mechanism of coagulation and main coagulation proteins, notably factor XIII, were elucidated. In 1975, Matras was the first to use fibrin adhesive properties through highly concentrated fibrinogen. Since then, the biological glues have definitely supplanted the synthetic glues and are increasingly used in human clinical practice.
The biological glues introduce a new approach to surgeries and sutures. Surgeons have sought for a long time an effective, easy-to-use and above all easily tolerated adhesive that could be used in addition to or in replacement of sutures. Surgical sutures are important nowadays. However, numerous problems arise such as intolerance or toxicity. Blood, through its coagulation properties, has always represented for surgeons an ideal model of biological gluing but the use of biological glues prepared from human source raises a viral transmission problem. Virus transmission hazards depend greatly on the purification methods of plasma concentrates. For human clinical use, the biological glues must be prepared with the severe treatments for viral inactivation without affecting the quality of the products. Research is still under way to develop an adhesive combining the following properties:
high viral safety PA1 sufficient adhesivity PA1 good elasticity PA1 good hold on adjacent tissues PA1 absence of toxicity PA1 absence of metabolic action PA1 good tolerance PA1 a) recovering the supernatant obtained from step a) of the method of claim 1; PA1 b) diafiltering said supernatant against an exchanged low or free salt solution; PA1 c) diluting said diafiltered supernatant until the obtention of a prothrombin solution of about 100 mosmoles/kg of weight or lower; PA1 d) precipitating prothrombin by adding an acidic solution until a pH of about 5.2 is obtained; PA1 e) solubilizing the precipitate of step d) in a solution having a near neutral pH; PA1 f) converting the prothrombin of step e) into thrombin in the presence of a diluting solution of calcium chloride to achieve a concentration of calcium chloride of about 20 to about 30 mM; PA1 g) incubating said thrombin with a viricide solvent/detergent solution in an amount sufficient to inactivate lipid-containing viruses; PA1 h) purifying the incubated material by sequential ion-exchange chromatography using a single sulfalkyl-activated polysaccharide cation exchange medium selected from the group consisting of a sulfakyl-activated polyagarose, a sulfakyl-activated polydextran and a noncompressible composite medium of sulfalkyl-activated dextran and silica particles having a high selectivity for thrombin using as an eluting agent at least three and increasing concentrations of an aqueous buffer solution; and PA1 i) recovering thrombin peak eluate from the chromatography of h) and exchanging the buffer of the eluate with a physiologically compatible stabilizing formulation buffer for stabilizing the recovered thrombin and recovering a formulation buffer solution of thrombin.
The U.S. Pat. Nos. 5,290,918 and 5,395,923 issued to Haemacure Biotech Corp. described the methods of preparation and use of a concentrate of fibrinogen, Factor XIII and fibronectin for therapeutic purposes.
Because of its coagulation properties, the concentrate rich in fibrinogen and Factor XIII provides clinicians with a precious and effective tool for surgery, where haemostatic properties are greatly needed. The fields of clinical applications may be: neurosurgery, cardiovascular surgery, plastic surgery (skin graft), ORL surgery, stomatology, orthopedic surgery, general surgery and traumatology.
The main protein in this concentrate is fibrinogen which, through an enzymatic reaction in presence of thrombin and calcium ions, produces fibrinopeptides A and B permitting the formation of fibrin monomers. These monomers polymerize quickly and become soluble fibrin. Then, the fibrin stabilizing factor (Factor XIII), under the agency of calcium ions forms covalent bonds with the dissolved fibrin, which make it stable and insoluble in an acidic medium or in the presence of urea.
The fibrin thus formed is irreversible, stable and fully plays its role as coagulant. It resists fibrinolysis because of its high concentration, plasminogen free fibrinogen and keeps its shape as a result of the absence of exudation. This concentrate has the following characteristics: excellent stability after being dissolved again in a aqueous solution, solubilization at room temperature in a few minutes, good elasticity and, lastly, a good adhesion.
These characteristics depend only on the method of preparation from plasma. This is a simple, quick method easily adaptable to industrial production. All the concentrate biological and biochemical are preserved, and the product meets clinical requirements.
The use of blood-borne products always posed viral transmission problems despite available virological tests. The safest way to provide blood-borne safe products is to systematically inactivate viruses suspected to be present using appropriate techniques without deteriorating the biochemical properties of the plasma products. Numerous methods of viral inactivation based upon the nature of the viruses and the type of the proteins to be isolated are currently known, which is reflected by the increasing body of scientific publication in this respect.
The most widely used plasma products are albumin, immunoglobulins and concentrates of coagulation factors. In 1948, Gellis et al. were the first to use a method of inactivating viruses by heating an albumin preparation at 60.degree. C. for ten hours. This method is currently used since that date due to its verified efficacy to reduce risks of viral transmission. The same method has been applied to the preparation of immunoglobulins G (IgG) with the same efficacy. This efficacy can be related to the method of purification of these blood products, particularly the use of a complete fractionation procedure as described by Cohn, or Kistler and Nitschmann.
The use of ethanol in numerous steps of fractionation of albumin and IgGs allows for a repartition of the quantity of viruses in different fractions. Ethanol is known as a disinfecting agent against pathogenic agents, such as viruses, as mentioned by Henin et al. (1988) and Morgenthaler (1989).
Pasteurization of albumin and IgGs appeared at the beginning of the 50s. This technique, however, was directed to inactivation of hepatitis virus (hepatitis B and non A-non B). Curran et al. (1984) raised the issue of viral transmission of HIV type by transfusion or the use of other blood derivatives, particularly coagulation factors. Since then, methods of viral inactivation focused on HIV. No HIV transmission was signaled from the use of albumin or IgGs, this lack of viral transmission being assigned to the step of pasteurization (Mitra et al. (1986)). Coagulation factors such as factor VIII and IX are widely used by hemophilic patients. Heimburger et al. (1980) have applied to these products the same pasteurization technique as described for albumin for inactivating viruses during the preparation of factor VIII in the presence of glycine and sucrose, in order to avoid proteic denaturation under thermal denaturation at 60.degree. C. for ten hours. Their studies demonstrated the efficacy of inactivation of HIV, hepatitis B and hepatitis non A-non B. Hilfenhaus et al. (1985, 1986) confirmed that pasteurization is an efficient method for inactivating viruses such as HIV during the preparation of a concentrate of factor VIII. Tabor et al. (1982) inactivated hepatitis B virus by heating antithrombin III in the presence of citrate as a stabilizing agent. Hollinger et al. (1984) heated a concentrate of factor VIII in a lyophilized state for reducing the risk of transmission of HIV and hepatitis. Piszkiewicz et al. (1988) demonstrated that heat treatment of lyophilized concentrates of coagulation factors did not have any significant effect on the activity of these factors. These authors stressed that it was not obvious to find any production of neoantigens due to heat treatment. Studies on viral inactivation by heat treatment were conducted by Piszkiewicz et al. in preparations of "anti-hemophilic factors" (Hemofil.RTM. T, Hemofil.RTM. CT), wherein the latter were heated during 72 hours at 60.degree. C., and during the preparation of anticoagulant inhibitor complexes (Autoplex.RTM. T), factor IX complex (Proplex.RTM. SX-T and Proplex.RTM.) T), wherein the latter were heated during 144 hours at 60.degree. C. There has been no report on HIV seroconversion due to the use of any of the five heat-treated coagulation products. Hemofil.RTM. T concentrate made from plasma which has been screened of HBsAg and anti-HBc has been found not to transmit NANBH in a simian study. However, use of the same product made from plasma screened only for HBsAg resulted in NANBH in patients (Colombo et al. 1985). Hemofil.RTM. T is currently manufactured from plasma which is nonreactive for HBsAg and has normal ALT levels.
Viruses as well as proteins are more stable and more resistant to heat when in a dry state (lyophilized). The temperature and the duration of heating appear to vary upon the nature of the proteins to minimize denaturation thereof. However, the efficacy of viral inactivation have never been reported as perfect: NANBH transmission has been signaled by Colombo et al. HIV transmission has been reported by White et al. (1986) and Van den Berg et al. (1986). For these reasons, Winkelman et al. (1985) heated concentrate of factor VIII (type 8Y), factor IX (type 9A), factor VII, factor XI and thrombin at 80.degree. C. for 72 hours. Studies conducted on 29 patients having received these heat-treated products have shown that there was no seroconversion of HIV of HB and that there was a significative reduction of the incidence of transmission of NANBH.
Other methods of viral inactivation have been developed using a light sources (UV, gamma rays, and laser) to irradiate the infectious agents in plasma. The following are cited as references: Oliphant et al.: Homologous serum jaundice: experimental inactivation of etiologic agent in serum by ultraviolet irradiation (Publ. Health Rep 1946; 61: 598-600), Wolf et al.: Ultraviolet irradiation of human plasma to control homologous serum jaundice (JAMA 1947; 135; 476477), Blanchard et al.: Methods of protection against homologous serum hepatitis. II. The inactivation of hepatitis virus serum with ultraviolet rays (JAMA 1948; 138:341), Murray et al. Effect of ultraviolet radiation on the infectivity of icterogenic plasma (JAMA 1955: 157: 8-14). Gurzadyan et al.: Mechanism of high power picosecond laser UV irradiation of viruses and bacterial plasmids (Photochem. Photobiol. 1981; 3: 835-838), Redfield et al.: Psoralen inactivation of influenza and herpes simplex virus and of viral infected cells (Infect Immun 1981; 32: 1216-1226), Heindrich et al.: Clinical evaluation of the hepatitis safety of beta-propiolactone-ultraviolet treated factor IX concentrate (PPBS), (Throm. Res. 1982; 28: 75), Kitchen et al.: Effect of gamma irradiation of the human immunodeficiency virus and human coagulation proteins (Vox Sang, 1989, 56: 233-229).
For more than ten years, one of the most currently used method for viral inactivation of viruses in blood-borne products is a method combining the use of a solvent and a detergent. This method has been developed by Neurath and Horowitz (U.S. Pat. No. 4,540,573 issued in September 1985, U.S. Pat. No. 4,613,501, U.S. Pat. No. 4,764,369 issued in August 1988; U.S. Pat. No. 4,820,805 issued in April 1989; U.S. Pat. No. 4,841,023 issued in June 1989 and U.S. Pat. No. 5,541,294 issued in July 1996). The mixture or solvent/detergent (Tri(n-butyl) phosphate/detergent) typically inactivates enveloped viruses such as HIV, HTLV-I, HBV and EBV.
Solvent/detergent method is however not sufficient to provide safe plasma products, because of the eventual presence of non-enveloped viruses such as parvovirus and poliovirus which are insensitive to solvent/detergent. Another technique has been recently introduced for eliminating non-enveloped viruses on which solvent/detergent has no effect. This technique is a nanofiltration. Nanofilters are composed with microporous fibers and have been commercialized under the name Planova BMM (Asahi Chemical Industries, Tokyo, Japan). The porosity of these filters varies from about 15 to 35 nm. These filters can retain certain types of viruses having a size larger than about 25 nm. These filters are efficient for eliminating viruses such as HIV-I (80-100 nm), HBB (42 nm), HCV (&lt;80 nm), hepatitis Delta virus (35 nm), bovine viral diarrhea virus (60-70 nm), Sindbis virus (60-70 nm), reovirus type 3 (60-80 nm), poliovirus Sabin type 1(25-30 nm), human parvovirus (20-25 nm); Sekiguchi et al.: Possibility of hepatite B virus (HBV) removal from human plasma using regenerated cellulose hollow fiber (BMM) (Membrane, 1989; 14: 253-261), Hamamoto et al.: A novel method for removal of human immunodeficiency virus: filtration with porous polymeric membranes (Vox sang., 1989; 56: 230-236), Tsurumi et al.: Structure of cuprammonium regenerated cellulose hollow fiber (BMM hollow fiber) for virus removal (Polym. J. 1990, 22: 751-758), Ishikawa et al.: Novel determination method of size of virus in solution using cuprammonium regenerated cellulose membrane (BMM) (Membrane, 1991; 16:101-111), Tomokiyo et al.: Studies on virus elimination and inactivation effect of highly purified F-VIII concentrate (The clinical report, 1991; 25: 271-275), Manabe: Virus removal and inactivation in process validation (Animal Cell Technology: Basic & Applied Aspects (Murakami, H., Shirahata, S., Tachibana, H. eds, 1992, 15-30), Burnouf et al.: Strategy of virus removal/inactivation of plasma-derived products: Interest of nanofiltration as a new virus elimination method (manuscript submitted to JAACT 93).
Rubinstein et al. used a double viral inactivation of factor VIII concentrate by treating the latter with solvent/detergent and heating at 100.degree. C. for 30 minutes the final product. Upon these authors, thermal treatment of the final product allows the inactivation of nonlipid-enveloped non A-non B hepatitis viruses. Heat treatment of the final product is also a cautious measure in case of accidental viral contamination during manipulation or due to the equipment (Vox Sang, 1991: 60: 60).
Recently, Proba et al. introduced a triple viral inactivation during the preparation of thrombin: (1) solvent/detergent treatment, (2) nanofiltration and (3) heat treatment at 100.degree. C. for one hour of the lyophilized product (U.S. Pat. No. 5,506,127 issued to Haemacure Biotech Inc.).
The triple viral inactivation treatment confers an increased safety in the use of blood-borne products, particularly coagulation factors or biological glue.
Nobody has described a three viral inactivation step process for preparing a concentrate of other coagulation factors such as fibrinogen. Furthermore, the viral inactivation steps may also mean that the addition of numerous steps in a process of making blood-borne products will lead to a diminution of recovery of useful products. There is still room to improve recovery of blood-borne products and this, not at the expense of viral safety and product quality, or to improve product safety without sacrificing the recovery and nature of the product.